Project Abstract The virulence of a bacterium is closely linked to its ability to release secreted factors. It follows that many evolutionarily distinct pathways exist for the translocation of proteins in pathogenic Gram-negative bacteria (types I-VI). Type VI secretion (T6S), the most recently described of these pathways, is an important contributor to the virulence of many pathogenic bacteria. The PI of this proposal, Dr. Joseph Mougous, first characterized this secretion system in Pseudomonas aeruginosa. This work produced fundamental biochemical and structural insights into the secretion system; furthermore, it provided evidence that the T6S system (T6SS) of P. aeruginosa is important for the devastating chronic infections this organism causes in cystic fibrosis patients. The focus of this proposal, and the major research emphasis of Dr. Mougous' laboratory, is to understand the regulatory pathway that tightly modulates the activity of the P. aeruginosa T6SS. This pathway, which is reminiscent of eukaryotic signaling, is initiated by dimerization of a transmembrane serine-threonine kinase. This provokes a series of events that triggers the secretion system, leading to substrate translocation. Although prior work by Dr. Mougous has revealed some of the mechanistic details of this pathway, many key questions remain unanswered. The proposed experiments seek to answer these mechanistic questions, and also to utilize this novel mode of regulation for the purpose of identifying substrates of the secretion system - a current impediment to the field. Many pathogens of significance to human health and biodefense require T6S for virulence and use this pathway to regulate its activity; therefore, findings generated by the proposed research will have broad implications and could lead to the development of new therapeutic strategies.